Microbiome Boot Camp

Utah Valley University - BIOL490R

Course goals

• Understand the data structure of microbiome studies
• Demonstrate how to process microbiome data into a usable format
• Explore processed microbiome data
• Test hypotheses
• Write a paper putting results into context of other research

Course requirements

• Laptop with R and R Studio installed
• Previous experience with R programming
  • data cleaning / transformations
  • plotting with ggplot
  • file management
  • model training and evaluation

Course setup

• We have a real data set and some question to address:
  • Pando foliar fungi
  • Are trees selecting fungal endophyte or epiphyte community?
  • Do foliar fungi follow stochastic assembly?
  • What spatial structure is there?

  • Hypotheses:

• We will use this data set to learn how to process and analyze microbiome data
• We will then write a paper as a class to present our findings for peer-review

Image from Wiki commons

“Pando is believed to be the largest, most dense organism ever found at nearly 13 million pounds. The clone spreads over 106 acres, consisting of over 40,000 individual trees. The exact age of the clone and its root system is difficult to calculate, but it is estimated to have started at the end of the last ice age. Some of the trees are over 130 years old. It was first recognized by researchers in the 1970s and more recently proven by geneticists. Its massive size, weight, and prehistoric age have caused worldwide fame.”

We are using Pando as a natural laboratory to study the biogeography and assembly of fungi associated with the leaves of plants. These fungi, known as endophytes (inside the leaves) and epiphytes (on surface of leaves) are important components of the plant microbiome. They modify plant disease severity, alter plant phenotype, and can even help plants resist common stressors such as drought. One important question regards where plants get their foliar fungi. In grasses, they are passed along inside seeds (vertical transmission), but in dicot plants like Pando, they are assembled from the environment.

They blow in on the wind, or are carried by animals, or by raindrops. But it is interesting that not all fungi can have a healthy stable relationship with all plants. There is likely some environmental filtering going on… the abiotic environmental variables or the plant itself are possibly doing some selecting of which fungi make it into plant leaves.

Most studies of this nature have to contend with the fact that when you go out into the field, each plant you sample has a different genotype, even if you sample the same plant species. We don’t have that problem in this study since every tree that’s part of Pando is just a piece of the same genetic clone individual.

What this means is that we can sample leaves from Pando and can study the spatial structure of the fungal communities without worrying about plant genotype.

Geoff Zahn, Josh Leon, Austen Miller inside Pando clone

In September 2023, we collected leaf samples from all over the Pando clone and extracted DNA from both the leaf surfaces and interiors. Those samples were sequenced on an Illumina MiSeq system (2x300bp). It’s there that we start our work. We will use bioinformatics tools to turn the raw DNA reads into fungal community data that we can work with and explore. We will test hypotheses about environmental filtering of foliar fungi.

Some questions we can ask:

• Are foliar fungi reflective of their immediate environment? i.e., Do samples that are physically close to each other share more similar fungal communities than can be expected by chance?
• Are there any “edge effects” in our communities? Are samples from the edge of the clone more similar to each other than those deeper inside the forest patch?
• Epiphytes might ‘just be there’ but endophytes are living inside the plant tissue. So do we see contrasting patterns between the two categories of fungi? If so, this could be evidence for environmental filtering of endophytes. And if there’s no geographic structure in endophytes, it could suggest that the plants themselves are doing the filtering.

Weekly tasks and assignments